This proposal focuses on the role of the membrane-lytic Membrane Attack Complex (MAC) in the pathology of Age-related Macular Degeneration (AMD), a debilitating disease that is a major cause of blindness. In humans, the MAC has been conserved through evolution, but its role now includes the destruction of sensitive host cells. Our prior and ongoing work on the high-resolution structures of MAC components, and the insights they provide into MAC assembly, place us in a unique position to propose and test mechanism-based approaches to discovering novel probes and inhibitors of MAC assembly. In order to approach these goals, we have formed a collaboration with Dr. Kumar-Singh at Tufts Medical School, who is an expert in murine models of AMD; and we have recruited Dr. Sergienko, an expert in high-throughput screening and assay development, who works in a major center for small-molecule discovery housed within the Sanford-Burnham campus (the Prebys Center). We have shown that MAC assembly requires conformational changes within each MAC component (C6-C9), switching from a compact auto-inhibited conformation to a highly extended state as it joins (and augments) the nascent pore. For Aim 1, we hypothesize that small molecules that bind to MAC proteins and stabilize their compact conformation could modulate this process and inhibit or promote pore formation. Using a small commercial screen and a high-throughput Protein Thermal Shift approach, we have already demonstrated feasibility. Thus, 12 out of 1280 compounds showed a significant ?TM (?1.5C), and we have tested 6 of these in a functional cell-based assay. Of the 6, we discovered 2 inhibitors and 2 activators of MAC pore formation, providing encouraging support for our hypothesis. We will perform a parallel study on C6, the first recruit to the MAC pore, for which we already have a crystal structure. In Aim 2, we will study binding on the macro-scale, using Isothermal Titration Calorimetry, and co-crystallization of the most promising compounds at 3 resolution or better, which will provide atomic models of protein with bound compounds. This will reveal the major determinants of binding and specificity, as well as strong clues into mechanism. Structural insights will feed back to Aim 1 to guide further screening, and forward to Aim 3 for selection of compounds for in vivo studies. In Aim 3, we will define the role of the MAC in a mouse model of AMD, using inhibitors and activators of MAC deposition characterized in Aims 1 and 2. The longer-term focus of this Aim will be on small-molecule inhibitors of MAC, as these could provide leads for novel therapeutics to tear AMD, thereby framing the next phase of our studies. The most efficacious compounds will feed back to Aims 1 and 2 to guide the selection of compounds for subsequent rounds of high-throughput screening, and help guide the selection of large-scale screens (~50,000) taken from our main ~700,000 compound library. We expect to discover small molecules with high potency and selectivity, and predict that the mechanistic insights and novel probes generated here will be relevant to other degenerative diseases in which the MAC is implicated.